Hybrid dairy cattle and systems for maximizing hybrid advantage

ABSTRACT

Systems for selecting, generating, and breeding hybrid dairy cattle are described, as are methods for maintaining herds of hybrid cattle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/169,459, filed Nov. 13, 2015, which is continuation of InternationalPatent Application No. PCT/US15/60576, filed Nov. 13, 2015, which claimsthe benefit of U.S. Provisional Application No. 62/106,151, filed Jan.21, 2015, and U.S. Provisional Application No. 62/080,145, filed Nov.14, 2014, of which each are incorporated herein by reference in theirentirety.

BACKGROUND

The bovine industry is highly specialized and dairy cattle have beenbred to efficiently produce large volumes of milk. The United Statesdairy herd produced 83.9 billion kg (185 billion lbs.) of milk in 2007,up from 52.6 billion kg (116 billion lbs.) in 1950, yet there are onlyabout 9 million cows on U.S. dairy farms—about 13 million fewer than in1950.

SUMMARY

The present invention provides technologies for generating and/ormaintaining a herd of high-quality hybrid (i.e., cross-bred) dairycattle. The present invention also provides technologies foridentification, characterization, and/or selection of dairy cattlebreeding stock sires/sire lines and dams/dam lines.

Among other things, the present invention encompasses the identificationof a source of a problem with traditional cross-breeding technologies,particularly those that select one or both of the sire/sire line ordam/dam line (referred to herein as male and female “F0 individuals” or“F0 lines”) based on performance characteristics of the F0 individualsor lines themselves. For example, it is particularly common in the fieldof dairy cattle breeding to select dams/dam lines based on themilk-production characteristics of particular dam individuals or damlines. The present disclosure encompasses the recognition that suchapproaches may actually fail to select F0 individuals or lines that aremost desirable for creating hybrid dairy animals.

Moreover, the present invention encompasses the recognition that, whilecross breeds display hybrid vigor and idealized traits based upongenetic selection, it is difficult using presently available strategiesto maintain and reproduce such traits among subsequent generationswithin the herd.

In some embodiments, the invention provides methods comprising steps of:assaying members of each of a plurality of F1 crossbreed cattle; andselecting as breeding stock F0 cattle based on the performance of the F1crossbreed cattle.

In some embodiments, selecting F0 cattle based on performance comprisesselecting based on level or nature of at least one performancecharacteristics for which the F1 cattle are superior to both F0s. Insome embodiments, the step of selecting cattle based on performancecomprises detecting a genetic signature correlated with the level ornature of the one or more performance characteristics.

In some embodiments, the F1 cattle are dairy cattle. In someembodiments, the F1 cattle are dairy cattle females. In someembodiments, the F1 cattle are beef-on-dairy cattle.

In some embodiments, the invention provides methods of breeding cattlecomprising: generating an embryo from male and female F0 gametes,wherein one or both of the male and female F0 gametes is from an F0individual selected based on performance of prior F1 progeny resultingfrom a cross of gametes from the F0 individual, wherein the male andfemale F0 gametes are from different breeds.

In some embodiments, the invention provides methods of generating an F1herd of animals comprising steps of: implanting into each of a pluralityof host female cattle, an F1 embryo resulting from fertilization of anF0 female gamete with an F0 male gamete, wherein one or both of the maleand female F0 gametes is from an F0 individual selected based onperformance of prior F1 individuals resulting from a cross of gametesfrom the F0 individual.

In some embodiments, the invention provides methods of maintaining an F1crossbreed herd of animals comprising steps of: implanting an F1crossbreed embryo into each of a plurality of F1 female dairy cattle,wherein each of the F1 female dairy cattle resulted from a first“F1-selected crossbreed cross”, wherein an “F1-selected cross” is onethat involves fertilization of an F0 female gamete with an F0 malegamete, wherein: a) the F0 male and female gametes are from differentbreeds; and b) one or both of the F0 male and female gametes is from anindividual or line selected performance of prior F1 progeny generated incrosses of the same individual or line; wherein each implanted F1crossbreed embryo results from a second F1-selected crossbreed cross.

In some embodiments, the invention provides methods of maintaining acrossbred herd of hybrid F1 animals possessing desired traits, whereinthe hybrid state of the F1 trait is maintained through embryonictransfer of embryos selected based on F1 performance.

In some embodiments, the second F1-selected crossbreed cross utilizesmale F0 gametes, female F0 gametes, or both male and female F0 gametesfrom the same individual or line utilized in the first F1-selectedcrossbreed cross. In some embodiments, the second F1-selected crossbreedcross utilizes male F0 gametes from the same individual or line utilizedin the first F1-selected crossbreed cross. In some embodiments, thesecond F1-selected crossbreed cross utilizes male F0 gametes from thesame individual in the first F1-selected crossbreed cross. In someembodiments, the second F1-selected crossbreed cross utilizes female F0gametes from the same individual or line utilized in the F1-selectedcrossbreed cross. In some embodiments, the second F1-selected crossbreedcross utilizes female F0 gametes from the line utilized in the firstF1-selected crossbreed cross.

In some embodiments, the second F1-selected crossbreed cross utilizes amale or female gamete from an individual or line different from thatutilized in the first F1-selected crossbreed cross. In some embodiments,the different individual or line is selected based on improvedperformance of its F1 progeny relative to those of the corresponding F0individual or line used in the first F1-progeny selected crossbreedcross.

In some embodiments, the invention provides a method further comprisingsteps of: assaying one or more performance characteristics of F1individuals resulting from a plurality of crosses that utilize F0gametes from one or both of the F0 individuals or lines utilized in thefirst F1-selected crossbreed cross; comparing the assayed one or moreperformance characteristics with those of F1 individuals resulting froma plurality of crosses that utilize F0 gametes from the different F0individual or line, so that the different individual or line is selectedas a desirable breeding stock individual or line.

In some embodiments, the invention provides herds of female cattle, eachof which is pregnant with an F1 embryo resulting from fertilization ofan F0 female gamete with an F0 male gamete, wherein one or both of themale and female F0 gametes is from an F0 individual selected based onperformance of prior F1 individuals resulting from a cross of gametesfrom the F0 individual.

In some embodiments, the invention provides herds of female cattlecomprising: a plurality of F1 animals, each of which was generated fromF1 embryo resulting from fertilization of an F0 female gamete with an F0male gamete, wherein one or both of the male and female F0 gametes isfrom an F0 individual selected based on performance of prior F1individuals resulting from a cross of gametes from the F0 individual.

In some embodiments, the invention provides F1 herds of female cattlecomprising: a plurality of F1 animals, each of which was generated fromF1 embryo resulting from fertilization of an F0 female gamete with an F0male gamete, wherein each male F0 gamete is from the same F0 individualand each female gamete is from a female within the same F0 family,wherein one or both of the F0 gametes is from an individual or familythat is selected based on performance of prior F1 individuals resultingfrom a cross of gametes from the F0 individual or from a member of theF0 family. In some embodiments, each member of the plurality ischaracterized by level of one or more performance parameters that issuperior to that of either F0.

In some embodiments, the invention provides pluralities of embryos, eachof which was generated by fertilization of an F0 female gamete with anF0 male gamete, wherein one or both of the male and female F0 gametes isfrom an F0 individual selected based on performance of prior F1individuals resulting from a cross of gametes from the F0 individual. Insome embodiments, the embryos are frozen after fertilization for futureimplantation. In some embodiments, the embryos are generated fromgametes of cross-bred F0 cattle.

In some embodiments, the invention provides methods of selecting an F0sire or sire line for use as breeding stock for dairy cattle, the methodcomprising steps of: assaying at least one performance attribute of atleast one first F1 progeny individual that results from a first cross,which is a cross of a first candidate F0 sire individual with aparticular F0 dam or dam line; comparing the assayed at least oneperformance attribute with that of at least one second F1 progenyindividual that results from a reference cross, which reference is: across of a reference F0 sire individual with the particular F0 dam ordam line; or a second cross of the particular F0 sire individual with asecond F0 dam or dam line, which may be the same or different from theparticular F0 dam or dam line; or a cross of at least one differentcandidate F0 sire individual with the particular dam or dam line; andselecting as an F0 stock sire or sire line an F0 sire candidate whose atleast one F1 progeny individual showed superiority in the comparing.

In some embodiments, the invention provides methods for selecting an F0dam or dam line for use as breeding stock for dairy cattle, the methodcomprising steps of: assaying at least one performance attribute of atleast one first F1 progeny individual that results from a first cross,which is a cross of a first candidate F0 dam or dam line with aparticular F0 sire individual; comparing the assayed at least oneperformance attribute with that of at least one second F1 progenyindividual that results from a reference cross, which reference is: across of a reference F0 dam or dam line with the particular F0 sire; ora second cross of the particular F0 dam or dam line with a second F0sire, which may be the same or different from the particular F0 sire; ora cross of at least one different candidate F0 dam or dam line with theparticular sire; and selecting as an F0 stock dam or dam line an F0 damor dam line candidate whose at least one F1 progeny individual showedsuperiority in the comparing.

In some embodiments, pluralities of F1 progeny from each cross areassayed.

In some embodiments, potential performance attributes comprise: milkproduction, longevity, semen production, age at first calving, bodydepth, cell counts, cow conception rate, dairy form, daughter calvingease, daughter pregnancy rate, daughter still birth, fat pounds, fatpercent, feet and legs score, fertility, final score, foot angle, foreudder attachment, front teat placement, heifer conception rate, ketosis,lameness rate and/or degree, locomotion, milk productive life, milkingspeed, protein percent, protein pounds, rear legs rear view, rear legsside view, rear teat placement, rear udder height, reproductive life,resistance to cold, resistance to disease (e.g., mastitis, metritis,etc.), rump angle, rump width, somatic cell score, sire calving ease,sire still birth, size, stature, strength, teat length, udder cleft,udder conformation, and udder depth.

In some embodiments, the invention provides methods of providing across-breed total performance index based on performance attributesassayed from F1 cattle. In some embodiments, a cross-breed totalperformance index embodied in computer-readable format. In someembodiments, the performance parameter is not shared by F0 individuals.In some embodiments, the performance parameter is superior in F1 ascompared with F0 individuals or both F0 parents.

In some embodiments, crosses are repeated over time to adjust theperformance characteristics of the F1 progeny.

In some embodiments, the invention provides methods of operating abusiness, the method comprising steps of: providing semen from aselected F0 stud for dairy cattle, which F0 stud is selected based onperformance of F1 progeny resulting from prior cross-breed crosses inwhich semen from the selected F0 stud fertilized F0 female gametes. Insome embodiments, the semen primarily includes sperm of one gender orthe other. In some embodiments, semen primarily includes “female” sperm.

In some embodiments, the invention provides methods comprising steps of:collecting performance information for F1 progeny resulting from each ofa plurality of crosses in which gametes from a first F0 individual arecrossed with gametes from at least one second F0 individual of oppositegender from the first F0 individual; storing the performance informationin a computer-readable format that permits its retrieval or analysislater in time. In some embodiments, steps of collecting performanceinformation comprises collecting performance information for a pluralityof different crosses in which gametes from the first F0 individual arecrossed with gametes from a plurality of different second F0individuals. In some embodiments, pluralities of different crosses areperformed at different points in time. In some embodiments, storedperformance information is compared with that from one or more crossesin which gametes from a second F0 individual of matching gender to thefirst F0 individual are crossed with gametes from at least one second F0individual of the opposite gender. In some embodiments, at least one ofthe crosses in which gametes from the matching gender second F0individual are crossed with gametes from at least one opposite gendersecond F0 individual, involved gametes from at least one opposite genderF0 individual who is the same as one with whom gametes from the first F0individual had been crossed.

In some embodiments, the invention provides methods comprising steps of:using In Vitro Fertilization (IVF) to produce cross-breed embryos offemale dairy cattle. In some embodiments, F1 cattle are crosses of F0sselected based on performance. In some embodiments, breeds forgenerating cross-breed embryos are selected from the following:Holstein-Friesian, Brown Swiss, Guernsey, Ayrshire, Jersey, Red & White,Milking Shorthorn, Linebackers, Dutch Belts, Burlina, Belarus Red,Belted Galloways, Canadienne, Carora, Danish Jersey, Frankeston Red,German black pied, Girolando, Illawarra, Meuse-Rhine-Issel, and Siboneyde Cuba.

In some embodiments, the invention provides methods comprising steps of:breeding dairy cattle to a cross-breed index.

In some embodiments, all of the F0 female gametes are from the same F0female individual, all of the F0 male gametes are from the same F0 maleindividual, or both.

In some embodiments, fertilization is performed in vitro. In someembodiments, fertilization is performed in vivo.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts conventional, natural-mating cross-breeding strategies.In particular, this Figure illustrates three-breed and two-breedrotation strategies that are employed in pursuit of hybrid vigor. As canbe seen with reference to this Figure, heterosis is maximum in the firstcross, and neither scheme achieves this level again. Neither of thestrategies depicted can generate and maintain herds of F1 animals (i.e.,of 50/50 NRF/H animals). Furthermore, complications arising fromcrossing over and/or segregation of chromosomes can result insignificant dis-uniformity in characteristics of progeny generated bythe serial matings in such strategies.

FIGS. 2A-D illustrate chromosomal segregation and crossing-over eventsthat occur during matings as depicted in FIG. 1. FIG. 2A depictsrepresentative chromosome pairs as found in somatic cells of an F1hybrid animal resulting from a Holstein and Jersey cross. F1 hybrids areuniform: each chromosome pair has a 50/50 contribution from the twofounder breed parents. Thus, the two founder breeds are represented inthe same proportion in the F1 genome. Cow to cow, the two founder breedsare represented in the same proportions on each chromosome. Moreover,each individual chromosome within a chromosome pair is 100% from onefounder strain. FIG. 2B illustrates representative chromosomal makeup ingametes of an F1 hybrid animal whose somatic chromosomes are depicted inFIG. 2A. As can be seen, cross-over events that occur during gameteproduction can result in individual chromosomes with differentpercentages of contributions from each of the two corresponding founderchromosomes. These cross-over events result in shuffling of encodedtraits, and therefore complicate efforts to predict how and which traitsare expressed in the next generation. FIGS. 2C and 2D illustrate thischallenge, specifically depicting results of mating gametes of FIG. 2Bwith gametes of a third breed. As shown in FIG. 2C, whether from asingle animal or different F1 animals, every egg will have a unique mixof haplotypes from the two breeds. On average they are uniform, but asindividuals they are each unique. For example, FIG. 2D illustrates 3different intended “replacement” individuals, who are progeny of the F1x third breed cross illustrated in FIGS. 2A-2C and who all have overallgenome contributions that are 25% founder strain 1, 25% founder strain2, and 50% third breed but clearly have completely different geneticmakeup and therefore have different traits and characteristics. Thiscomplexity increases in subsequent crosses, so that it is virtuallyimpossible to maintain F1 hybrid traits through such conventional matingstrategies.

FIG. 3 outlines an inventive system for providing valuable valuedproducts and services to dairy customers based upon embodiments of thepresent disclosure. Components of the depicted system include: 1) a twoline breeding system; 2) a testing system; 3) an IVF and ET transferservice; and 4) a sales force.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are defined below. Those skilled in the art willappreciate that definitions for certain terms may be provided elsewherein the specification, and/or will be clear from context.

Allele: As used herein, the term “allele” refers to one of two or moreexisting genetic variants of a specific polymorphic genomic locus.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Artificial Insemination (AI): As used herein, the term “artificialinsemination (AI)” refers to introduction by the hand of man of semeninto a female bovine's uterus to achieve pregnancy. In many embodiments,AI is utilized in breeding, for example so that resulting pregnanciesare (or are intended to be) carried to term. In some embodiments, AI iscarried out with collected semen. In some embodiments, AI is carried outwith extracted semen. In some embodiments, AI is carried out with sementhat has been processed; for example, in some embodiments, the semen hasbeen sexed so that it is enriched for sperm of only one gender.

Those skilled in the art will appreciate that unless otherwise expresslyindicated, the term “AI” does not encompass embryos transfer procedures,where, for example, semen may be introduced into a cow to generateembryos for transfer.

Autosome: As used herein, the term “autosome” refers to any chromosomeexclusive of the X and Y sex chromosomes.

Breed: As used herein, the term “breed” refers to a group of cattlehaving common ancestors and/or sharing certain distinguishable traitsthat are not shared cattle of other breeds. Those skilled in the art arefamiliar with breed standards and/or characteristics. In manyembodiments, a particular breed is produced and/or maintained by matingparticular identified parent or parents (e.g., a particular sire with aparticular dam or with any one dame from of a particular dam line) withone another.

Chromosome: As used herein, the term “chromosome” refers to a linearmolecule of DNA with associated proteins in the nucleus of eukaryoticcells that carries the genes and functions in the transmission ofhereditary information.

Comparable: The term “comparable” is used herein to describe two (ormore) sets of conditions, circumstances, individuals, or populationsthat are sufficiently similar to one another to permit comparison ofresults obtained or phenomena observed. In some embodiments, comparablesets of conditions, circumstances, individuals, or populations arecharacterized by a plurality of substantially identical features and oneor a small number of varied features. Those of ordinary skill in the artwill appreciate that sets of circumstances, individuals, or populationsare comparable to one another when characterized by a sufficient numberand type of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder or with different sets of circumstances, individuals, orpopulations are caused by or indicative of the variation in thosefeatures that are varied. Those skilled in the art will appreciate thatrelative language used herein (e.g., enhanced, activated, reduced,inhibited, etc.) will typically refer to comparisons made undercomparable conditions.

Crossbreed: As used herein, the term “crossbreed” refers to cattleproduced from gametes of individual bovines that are different breeds orvarieties of cattle. Crossbreeding is often performed in dairy cattlefarming to produce healthier, more productive cattle compared to theparent breeds. Crossbreeding is the deliberate mating of animals fromdifferent breeds or strains; in many embodiments crossbreeding isdesigned to take advantage of heterosis (hybrid vigor) forcharacteristics like production, fertility, and longevity. In someembodiments, the present disclosure encompasses the insight that recentdevelopments relating to artificial insemination and/or in vitrofertilization, not typically employed in the dairy cattle industry, canbe utilized to enable and/or provide certain advantages with respect togenerating and/or maintaining crossbreed lines of dairy cattle asdescribed herein. As described herein, crossbreed cattle of particularinterest are hybrid cattle, in which 50% of the cattle's somaticchromosomes are from one strain or line and 50% are from a differentstrain or line (i.e., formed by crossing F0 individuals from first andsecond strains/lines that differ from one another. Those of ordinaryskill in the art will appreciate, however, that the term “crossbreed”can be used in some embodiments (as is clear from context) to refer toany individual whose genome, as a result of crossing, is not 100% fromany single breed. Diploid Cell: As used herein, the term “diploid cell”refers to a cell with a homologous pair of each of its autosomalchromosomes, with two copies (2n) of each autosomal genetic locus.

Embryo: As used herein, the term “embryo” refers to a fertilized oocyte(egg) prepared for immediate implantation within a female cattle orstored for eventual implantation within a female cattle.

F1: As used herein, the term “F1” refers to progeny cattle produced bycrossing two F0 individuals from different breeds or lines of cattle. F1cattle are also referred to as “hybrids,” and are characterized in that50% of their somatic chromosomes are from a first strain or line (i.e.,that of one of the F0 individuals), and 50% are from a second strain orline (i.e., that of the other F0 individual), different from the first.

F0: As used herein, the term “F0” refers to parental cattle who arecrossed to generate F1 hybrid cattle offspring.

Gametes: As used herein, the term “gametes” is used to refer toreproductive cells (e.g., spermatozoa or oocytes) having the haploidnumber of chromosomes, especially a mature sperm or egg capable offusing with a gamete of the opposite sex to produce a fertilized egg.Gametes are produced through the process of meiosis.

Gene: As used herein, the term “gene” refers to a DNA sequence in achromosome that codes for a product (e.g., an RNA product and/or apolypeptide product). In some embodiments, a gene includes codingsequence (i.e., sequence that encodes a particular product); in someembodiments, a gene includes non-coding sequence. In some particularembodiments, a gene may include both coding (e.g., exonic) andnon-coding (e.g., intronic) sequence. In some embodiments, a gene mayinclude one or more regulatory elements that, for example, may controlor impact one or more aspects of gene expression (e.g.,cell-type-specific expression, inducible expression, etc.).

Genome: As used herein, the term “genome” refers to the total geneticinformation carried by an individual organism or cell, represented bythe complete DNA sequences of its chromosomes.

Genome Profile: As used herein, the term “genome profile” refers to arepresentative subset of the total information contained within agenome. Typically, a genome profile contains genotypes at a particularset of polymorphic loci. In some embodiments, a genome profile maycorrelate with a particular feature, trait, or set thereofcharacteristic of, for example, a particular animal, line, breed, orcrossbreed population.

Genotype: As used herein, the term “genotype” refers to the diploidcombination of alleles at a given genetic locus, or set of related loci,in a given cell or organism. A homozygous subject carries two copies ofthe same allele and a heterozygous subject carries two distinct alleles.In the simplest case of a locus with two alleles “A” and “a,” threegenotypes can be formed: A/A, A/a, and a/a.

Genotyping: As used herein, the term “genotyping” refers to anexperimental, computational, or observational protocol fordistinguishing an individual's genotype at one or more well-definedloci. Those skilled in the art will be aware of a variety oftechnologies that can usefully and effectively perform genotyping. Insome embodiments, genotyping involves direct detection of a nucleic acidor nucleic acid sequence. In some embodiments, genotyping involvesindirect detection of a nucleic acid or nucleic acid sequence, forexample through detection or analysis of a proxy marker or event thatcorrelates with presence of the nucleic acid or nucleic acid sequence.

Haploid Cell: As used herein, the term “haploid cell” refers to a cellwith a single set (1n) chromosome of chromosomes—half the number of asomatic cell.

Heifer: As used herein, the term “heifer” refers to female cattle whohave not yet produced any calves.

Hybrid: As used herein, the term “hybrid” refers to cattle produced as aresult of crossing male and female gametes from different breeds orlines of cattle. Thus, typically, 50% of the autosomal genome (e.g., thesomatic genome) of a hybrid is from a first breed/line, and 50% is froma second breed/line. Of particular interest, as described herein, arehybrids in which 50% of its somatic chromosomes are from a first breedand 50% are from a second breed.

In Vitro Fertilization (IVF): As used herein, the term “in vitrofertilization” refers to a method of fertilizing an egg outside ofliving cattle. IVF is a process by which an egg is fertilized by spermoutside the body (i.e., in vitro, which literally translates to “inglass” but is understood in the art to refer to processes performed, forexample, in a laboratory or other artificial setting). In someembodiments, an IVF process may involve monitoring and/or stimulating afemale's ovulatory process, removing oocyte or oocytes (egg or eggs)from a female's ovaries, and/or contacting sperm and oocytes with oneanother in a laboratory (e.g., in a fluid medium) to achievefertilization. In some embodiments, IVF involves culturing a fertilizedegg (zygote) in a growth medium and/or either implanting it in afemale's uterus or storing it for future analysis and/or implantation.In some embodiments, IVF may involve sorting fertilized eggs forparticular desired attributes (e.g., gender).

Line: As used herein, the term “line” refers to a strain of cattledescended from common ancestral parents developed and maintained byselective breeding.

Mating: The term “mating,” as used herein, refers to a process thatresults in formation of an embryo, typically from two opposite-gendergametes. In some embodiments, mating involves natural service. In someembodiments, mating involves artificial insemination. In someembodiments, mating involves IVF. In many embodiments described herein,mating is utilized to generate hybrid progeny. In many embodiments,mating is utilized to generate crossbreed progeny.

Natural Service: As used herein, the term “natural service” refers totraditional cattle breeding of pairing males and females withoutartificial insemination or IVF-based techniques.

Phenotype: As used herein, the term “phenotype” refers to a trait, or toa class or set of traits displayed by a cell or organism. In someembodiments, a particular phenotype may correlate with a particularallele or genotype. In some embodiments, a phenotype may be discrete; insome embodiments, a phenotype may be continuous.

Single Nucleotide Polymorphism (SNP): As used herein, the term “singlenucleotide polymorphism” or “SNP” refers to a particular base positionin the genome where alternative bases are known to distinguish oneallele from another. In some embodiments, one or a few SNPs and/or copynumber polymorphisms (CNPs) is/are sufficient to distinguish complexgenetic variants from one another so that, for analytical purposes, oneor a set of SNPs and/or CNPs may be considered to be characteristic of aparticular variant, trait, animal, line, breed, cross-breed, or setthereof. In some embodiments, one or a set of SNPs and/or CNPs may beconsidered to define a particular variant, trait, animal, line, breed,cross-breed, or set thereof.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Trait: As used herein, the term “trait” refers to a detectable attributeof an individual. Typically, expression of a particular trait may befully or partially influenced by an individual's genetic constitution.In some embodiments, a trait is characteristic of a particularindividual, line, breed or crossbreed, for example in that it can berelied upon (individually or as part of a set) to distinguish thatindividual, line, breed, or crossbreed from others.

Detailed Description of Certain Embodiments

Dairy Cattle

Remains of domesticated cattle dating to 6,500 B. C. have been found inTurkey and other sites in the Near East approach this age also. Someauthorities date the domestication of cattle as early as 10,000 yearsago, and others almost half that amount of time. Early cattle served atriple-purpose. They provided meat, milk, and labor to their owners.Eventually their labor/draft purposes were largely replaced by horsesand machinery so they were selected more for single or in some casesdual purposes (milk and meat).

The Ayrshire breed originated in the County of Ayr in Scotland, prior to1800. Ayrshires are medium-sized cattle and should weigh over 1200pounds at maturity. They are strong, rugged cattle that adapt to allmanagement systems including group handling on dairy farms with freestalls and milking parlors. Ayrshires excel in udder conformation andare not subject to excessive foot and leg problems. Few other breeds canmatch the ability of the Ayrshire to rustle and forage for themselvesunder adverse feeding or climatic conditions. Ayrshire cattle will dobetter under pasture conditions than will the other major dairy breedsand, when pastures are poor, they need less grain to keep them in aircondition (C. H. Eckles, Dairy Cattle and Milk Production, 1923). Theruggedness of the terrain and the unfavorable climatic conditions oftheir native land led to the selection for those points of hardinessthat adapt them to less than ideal conditions. These traits makeAyrshires outstanding commercial dairy cattle.

The Ayrshire is a moderate butterfat breed. The actual average of allAyrshires on the official Dairy Herd Improvement Registry (DHIR) test isover 12,000 pounds of milk per year with a 3.9% test. Ayrshires respondto good management and feeding practices and individual Ayrshire herdsaverage as high as 17,000 pounds of milk per year and 700 pounds ofbutterfat. Top producing Ayrshires regularly exceed 20,000 pounds ofmilk per year in their lactations.

Other traits that make Ayrshires attractive to the commercial dairymaninclude the vigor of Ayrshire calves. They are strong and easy to raise.Ayrshires do not possess the yellow tallow characteristic that wouldreduce carcass value, so Ayrshire bull calves can be profitably raisedas steers.

The Guernsey cow is known for producing high-butterfat, high-proteinmilk with a high concentration of beta-carotene. Being of intermediatesize, Guernseys produce their high quality milk while consuming 20 to 30percent less feed per pound of milk produced than larger dairy breeds.They are also known for having a lower projected calving interval andhave a younger average age of first calf heifers than the larger breeds.Other attractive characteristics of Guernseys are their lack of anyknown undesirable genetic recessives and their adaptability to warmerclimates.

The Guernsey is also an excellent grazer. The cow is made forpasture-based milk production. Because of its grazing abilities, gentledisposition, calving ease, and ability to efficiently produce milk withless feed than other breeds, it is the ideal candidate for intensivegrazing. Dairy producers can realize profit potential while reducingmanagement costs.

Data from herds enrolled in the American Guernsey Association's DHIRprogram during 1992 shows the breed average to be 14,667 pounds of milk,659 pounds of butterfat and 510 pounds of protein per year on amature-equivalent basis.

Genetically, the Guernsey of today is much different than that of 960 A.D. Due to the advent and commercialization of artificial insemination, aprocess by which semen is introduced into a dam by other than naturalmeans, a particular bull can sire thousands of offspring. This geneticimprovement has been generated by a progressive, aggressive young sireprogram. Young bulls' semen is distributed throughout the Guernseypopulation until the bulls have a large enough daughter population thattheir offsprings' qualities are predictable. As proven bulls, thesesires may have as many as 1,500 daughters in up to 400 herds. However,every six months the list of available sires is updated. At that time,new bulls with superior genetics are added and older sires lose their“active” status. This insures that the breed-wide effort to improve theGuernsey's sound genetic base continues.

The Holstein cow originated in Europe. The major historical developmentof this breed occurred in what is now the Netherlands and morespecifically in the two northern provinces of North Holland andFriesland which lay on either side of the Zuider Zee. The originalstocks were the black animals and white animals of the Batavians andFriesians, migrant European tribes who settled in the Rhine Delta regionabout 2,000 years ago. For many years, Holsteins were bred and strictlyculled to obtain animals which would make best use of grass, the area'smost abundant resource. The intermingling of these animals evolved intoan efficient, high-producing black-and-white dairy cow.

A healthy Holstein calf weighs 90 pounds or more at birth. A matureHolstein cow weighs about 1500 pounds and stand 58 inches tall at theshoulder. Holstein heifers can be bred at 15 months of age, when theyweigh about 800 pounds. It is desirable to have Holstein females calvefor the first time between 24 and 27 months of age. Holstein gestationis approximately nine months. While some cows may live considerablylonger, the normal productive life of a Holstein is six years. Averageproduction for all Holsteins enrolled in official U.S.production-testing programs in 1987 was 17,408 pounds of milk, 632pounds of butterfat and 550 pounds of protein per year.

The Jersey breed originated on the Island of Jersey, a small Britishisland in the English Channel off the coast of France. The Jersey is oneof the oldest dairy breeds, having been reported by authorities as beingpurebred for nearly six centuries. The breed was known in England asearly as 1771 and was regarded very favorably because of its milk andbutterfat production.

Adaptable to a wide range of climatic and geographical conditions,outstanding Jersey herds are found from Denmark to Australia and NewZealand, from Canada to South America, and from South Africa to Japan.They are excellent grazers and perform well in intensive grazingprograms. They are more tolerant of heat than the larger breeds. With anaverage weight of 900 pounds, the Jersey produces more pounds of milkper pound of body weight than any other breed. Most Jerseys produce farin excess of 13 times their bodyweight in milk each lactation.

The American-type Jerseys were noted much more for production than forbeauty. Cattle referred to by this description are usually larger, a bitcoarser, and have been bred for years for those qualities that suit themfor milk and butterfat production. Additional emphasis on milkproduction and less stress on butterfat production had, no doubt,resulted in general acceptance of Jersey cows with more size and scale.Recent importations of Jerseys have consisted of larger cattle than manypreviously brought to the United States. Their offspring have not onlybeen acceptable in type but have also been used advantageously inimproving production.

One of the oldest recognized breeds in the world, Shorthorn cattleoriginated in Northeastern England in the Valley of the Tees River. TheMilking Shorthorn breed is the most versatile of all breeds and this isone of its greatest attributes. These docile cows efficiently producelarge volumes of nutritious milk each lactation and are large enough tohave a high salvage value when their long productive lives finally cometo an end. In addition, their healthy calves born each year on regularcalving intervals are spunky at birth, grow rapidly, and those not keptfor breeding stock and herd replacement make efficient gains and hangvery desirable grading carcasses. Other attributes of the breed includeease of calving, ease of management and economy of production,especially on home produced roughages and grass.

Belarus Red, also known as Byelorussian Red, Krasnaya belorusskaya, andKrasnobelorusskaya, is a Russian dairy cattle breed. The breed has beenimproved by crossing with Angeln Red, German Red, Polish Red, DanishRed, Estonian Red, and Latvian Brown. They are common in Belarus, mostlyaround Grodno and Minsk. They are noted for their longevity andundemanding feeding requirements.

The appearance of the Belarus Red cows is characterized by the followingfeatures. The head is medium long, not wide, with a long face. The pollis pronounced. The horns are of medium size. The neck is thin and ofmoderate length. The withers are not sharp, occasionally divided. Thechest is of medium depth, wide enough. The back is level, slightlynarrow. The loin is long and level, of medium width. The mid-part of thebody is well developed. The abdomen is capacious, not drooping. The rumpis level, slightly raised. The hindquarters are of medium length andwidth, with protruding hips. The legs are comparatively thin, bony, notlong, correctly set. Sometimes legs are splayed or bowed. The udder ismedium in volume, glandular, cup-shaped or roundish. The teats arecylindrical, of medium size. The skin is thin, elastic, mobile. Theskeleton is light and strong. The musculature is moderately developed.The conformation is harmonious and compact; the constitution delicate.

Many consider that the potential of the modern Belarus Red cattle hasnot been completely realized. The average milk yield of Belarus Redstock evaluated at the breeding farms in 1981 was 2557 kg with 3.69%fat; at the best farms it was 3053 kg with 3.62% fat. In the herd of theVasilishkovski breeding center in the Grodno region the average milkyield in 1982 was 2507 kg with 3.73% fat. In recent years several groupsof cows of this herd and of the Shchuchin experimental station averaged4514 kg of milk with 4.08% fat. In her 4th lactation cow Vyetv 2016produced 5986 kg of milk with 3.91% fat and 3.70% protein; Maltaproduced in her 3rd lactation, 6056 kg of milk with 4.55% fat; Volnaproduced, in her 4th lactation, 5906 kg with 4.85% fat.

Beef and fattening qualities of Belarus Red cattle are satisfactory:under favorable conditions of feeding and management the young stockdisplay a high growth rate and early maturity. The breed comprises 6basic lines and 2.'D families. To preserve these cattle conservationherds have been set up and a bank of frozen semen of the best sires ofall basic lines has been established. The breeding program for theimprovement of Belarus Red cattle aims at the following parameters forthe purebreds: the live weight of mature cows should be 500-540 kg, themilk production per lactation should be 4.5-5.0 thousand kg with fatcontent of 4.0-4.2% and protein content not less than 3.6%.

The Belted Galloway is a heritage beef breed of cattle originating fromGalloway in the west side of southern Scotland, adapted to living on thepoor upland pastures and windswept moorlands of the region. The exactorigin of the breed is unclear although it is often surmised that thewhite belt that distinguishes these cattle from the native blackGalloway cattle may be as a result of cross breeding with DutchLakenvelder belted cattle. It is the belt that gives them their name.Belted Galloways (or Belties) are primarily raised for their qualitymarbled beef, although they are sometimes milked and purchased to adornpastures due to their striking appearance.

Galloway cattle are naturally polled. Particularly visiblecharacteristics of the Belted Galloway are its long hair coat and thebroad white belt that completely encircles the body. Its coarse outercoat helps shed the rain, and its soft undercoat provides insulation andwaterproofing, enabling the breed to happily overwinter outside. BlackBelties are most prominent, but Dun and Red Belties are also recognizedby breed societies, the latter being comparatively rare and soughtafter. A female Belted Galloway cannot be registered in the Herd Book ifit has white above the dewclaw other than the belt, but can beregistered in the Appendix. A bull can only be registered in the Herdbook if it has no other white than the belt.

Bulls weigh from 1,700 pounds (770 kg) to 2,300 pounds (1045 kg) withthe average being 1,800 pounds (820 kg). Cows weigh from 1,000 pounds(450 kg) to 1,500 pounds (675 kg) with the average being 1,250 pounds(565 kg). Calves generally weight from 40 pounds to 60 pounds.

Belties are generally of a quiet temperament, but still maintain astrong maternal instinct and will protect a calf against perceivedthreats. Belties are well-suited for rough grazing land and will utilizecoarse grasses other breeds would shun. They are able to maintain goodcondition on less than ideal pasture, and produce a high quality beefproduct on grass alone. The USDA Cycle IV Germ Plasm Evaluation Programat the Meat Animal Research Center (MARC) showed that Galloway crossesplaced at the top of the chart for flavor, juiciness, and tendernesswhen compared to eleven other breeds.

Brown Swiss is a breed of dairy cattle that produces the second largestquantity of milk per annum, over 9,000 kg (20,000 lb.). The milkcontains on average 4% butterfat and 3.5% protein, making their milkexcellent for production of cheese. The Brown Swiss is known for a longgestation period, immense size, large furry ears, and an extremelydocile temperament. Regardless, the Brown Swiss is quite a resilientbreed of cattle; they are hardy and capable of subsisting with littlecare or feed.

The Brown Swiss originated on the slopes of the Alps in Switzerland;because they were bred in this harsh climate, they are resistant to theheat, cold and many other common cattle problems.

Norwegian Red (Norwegian: Norsk rødt fe) is a breed of dairy cattledeveloped in Norway. Often shortened to simply NRF, it has a red andwhite or black coat. Norwegian Reds are noted for their hardiness andthe richness of their milk.

Norwegian Red (NRF) is a dairy breed that has been selected for a broadbreeding objective, with increasing emphasis on functional traits likehealth and fertility. NRF—Norwegian Red was developed in the 1960sthrough crosses of dairy breeds with several Scandinavian breeds,including the Norwegian Red-and-White, Red Trondheim, and the Red PolledOstland. By the mid-1970s it became the dominant breed in its nativecountry, comprising 98% of the cattle population. Semen is frequentlyalso exported to North America for crossbreeding with Holstein cattle inthe U.S. dairy industry. Geno Breeding and A. I. Association, acooperative organization owned by Norwegian dairy farmers, is thebreeding organization for the Norwegian Red.

Production in the best herds exceeds 10,000 kilograms (22,000 lb.), withthe top cows milking more than 16,000 kilograms (35,000 lb.). Growthtraits are also included in the index, and young sires for progenytesting have a growth rate of approximately 1.4 kg/day. Fully-grown cowshave a live weight of up to 600 kilograms (1,300 lb.).

Health traits have been included in the net merit index since 1978.Currently mastitis and other diseases (in particular ketosis) areincluded in the breeding program. Although these are low heritabilitytraits, progeny testing based on a high number of daughters provides aselection index with high accuracy. Progeny testing for mastitis iscurrently based on approximately 300 daughters.

Norwegian Reds may be either polled or horned. Currently 50% of thecalves in Norway are born polled (genetically without horns). Systematicselection of polled sons after polled elite sires during recent yearshave increased the frequency of polled animals. It is expected thatNorwegian Red (NRF) breed will become a polled breed within the next20-25 years.

Conventional Dairy Cattle Husbandry

Animal husbandry is the management and care of farm animals by humansfor profit, in which genetic qualities and behavior, considered to beadvantageous to humans, are further developed. The term can refer to thepractice of selectively breeding and raising livestock to promotedesirable traits in animals for utility, sport, pleasure, or research.

Animal husbandry combines the art and science of raising animals byblending time-honored practices and modern scientific knowledge into asystem that provides for animal well-being and provides for safe andefficient management and handling of animals. Animal husbandry practiceschange as scientists, agricultural experts, and others involved withanimals learn new techniques or phase out those that are no longernecessary or appropriate. Animal husbandry practices range fromdehorning cattle to prevent injury to herd-mates and farm hands tomethods for housing livestock, providing adequate nutrition, anddevising breeding strategies.

Techniques such as artificial insemination and embryo transfer have beendeveloped and can be used to facilitate breeding. For example, becausesuch technologies permit a dam to carry an embryo other than her own,they can be used to ensure that large numbers of embryos from aparticular high quality dam (or dam line) can be implanted into alower-quality surrogate, thereby expanding the number of progeny thatcan be generated from the high-quality dam. This practice can vastlyincrease the number of offspring which may be produced by a smallselection of the best quality parent animals. However, as discussedherein, such technologies have not typically been employed with dairycattle. Among other things, they are often deemed to be too expensive towarrant use with dairy cattle. Also, to the extent that they tend toamplify particular genetic traits within a herd, they decrease geneticdiversity within the herd, increasing the severity of certain diseaseoutbreaks among other risks. Among other things, the present inventionencompasses the insight that such techniques, particularly when combinedwith crossbreeding strategies, can provide significant advantages in thehusbandry of dairy cattle as compared with conventional approaches.

In general, the present disclosure encompasses the recognition thatpresent day dairy farming has become a high risk business with very lowreturns on invested money. There is very little room for mistakes orextra expenses. Among other things, the cost of replacement females fora cow-calf operation is significant. Selecting replacement females ischallenging, especially when you consider that decisions made now willimpact your operation for many years. Therefore, dairy farmers have tofind ways to be as efficient with expenses and resources as possible.Ideally, dairy farmers need to maximize milk production and find ways toguarantee stable, high milk production from generation to generation.

Conventional Selection of Breeding Stock

In conventional animal husbandry approaches to breeding dairy cattle,dairy producers have a multitude of informational inputs available tomake sire selection decisions. For example, predicted transmittingabilities (PTAs) can be computed for various traits, for example in thebroad categories of production (milk and milk components),health/fitness, and type. Also, every 3 months, the Animal ImprovementPrograms Laboratory (AIPL) of the United States Department ofAgriculture releases the newest USDA-DHIA (Dairy Herd ImprovementAssociation) genetic evaluations for dairy bulls and cows. Dairy cattleare evaluated for the traits of milk, fat, and protein yield, length ofproductive life, and somatic cell score (an indicator of mastitis).Evaluation procedures combine information from all known femalerelatives of an evaluated animal, and from the animal itself in the caseof cows. Additionally, numerous type or conformation traits areevaluated routinely. The AIPL calculates genetic evaluations for typefor various breeds, and many breed associations provide their ownindexes or other strategies for evaluating certain breed-relevanttraits.

Traits are typically combined into an index based on their relativeeconomic weights. For example, the Net Merit index (NM$) computed byUSDA AWL estimates lifetime profit based on incomes and expensesrelevant for today's dairy producers and is expressed as a dollar value.Traits included in NM$ are: protein (lb.), fat (lb.), productive life,somatic cell score, udder composite, feet/legs composite, body sizecomposite and daughter pregnancy rate. Calving ability also is includedin NM$ calculations for Holsteins and Brown Swiss. The traitsincorporated into calving ability for Holsteins are daughter stillbirth,service sire stillbirth, daughter calving ease, and service sire calvingease. Only the two calving ease traits are available for inclusion incalving ability values of Brown Swiss.

Selection indexes developed by the various dairy breed associationstypically reflect genetic goals determined by their respective boards ofdirectors. The U.S. Ayrshire Breeders' Association uses a ProductionType Index (PTI) as a ranking tool for Ayrshire bulls. This indexaccounts for protein, fat, type, daughter pregnancy rate, udder depthand somatic cell score. The Progressive Performance Ranking (PPR) is theselection index used by the Brown Swiss Association. Traits included inthe PPR are protein, fat, somatic cell score, productive life, foot andleg composite, udder composite and daughter pregnancy rate. Theselection index developed by the American Guernsey Association is theProduction Type Index (PTI). Traits combined in the PTI are: protein,fat, type, udder composite, foot and leg composite, productive life,daughter pregnancy rate, somatic cell score, and strength. HolsteinAssociation USA calculates the Total Performance Index (TPI). Itincludes the traits of protein, fat, type, udder composite, feet and legcomposite, daughter pregnancy rate, productive life, somatic cell score,daughter calving ease, daughter stillbirth and dairy form. The JerseyPerformance Index, which is used by the American Jersey CattleAssociation, is comprised of the following traits: protein, fat,functional trait index, productive life, somatic cell score, anddaughter pregnancy rate. Functional trait index is based on the bull/cowPTAs for all type traits.

Using a selection index can be an effective way to consider severaltraits when choosing breeding stock. Conventional animal husbandrystrategies often rely on selection indexes, particularly for choosingservice sires.

Conventional Sire Selection Strategies

Breeding stock sires and sire lines are typically chosen based upontheir size and fertility. Prior successes in mating as well as siringfemales are both traits that are often utilized in selecting siresand/or sire lines.

Also, knowing where service sires rank relative to other active bulls istypically considered to be helpful in determining if the sires meet aparticular herd's genetic goals. Selection indexes can be particularlyuseful in monitoring such ranking. To maximize genetic improvement usinga selection index, it is usually recommended that the service sires fora given herd average at or above the 80th percentile.

Under conventional sire selection principles, sires of males (SM)represent the most elite males that are selected to be sires of the nextgeneration of young bulls. This group is chosen based on estimatedbreeding value (EBV) or genomic estimated breeding value (GEBV), and istypically composed of <5% of the males whose semen is marketed to dairyfarmers. These bulls are often referred to as “sires of sons.”

In accordance with these same conventional sire selection principles,sires of females (SF) represent a larger group of males that have beenselected based on EBV or GEBV and whose semen is used to breed thegeneral population and produce replacement females for commercial farms.These bulls are typically referred to as “active AI sires.”

Conventional Dam Selection Strategies

In conventional animal husbandry approaches to breeding dairy cattle,breeding stock dams and dam lines are typically chosen based upon theirmilk production capabilities and their fertility.

Under conventional dam selection principles, dams of males (DM)represent a group of elite females that are selected based on EBV orGEBV and that usually rank among the top 1% of the population. Thesecows are typically mated to elite bulls from the SM group for thepurpose of producing bull calves, and they are more commonly referred toas “bull mothers.”

In accordance with these same conventional dam selection principles,dams of females (DF) represent the large population of females that areprimarily used to produce milk rather than breeding stock. These cows,which are often referred to as “commercial cows,” are routinely mated tobulls from the SF group to initiate lactation, resulting in the nextgeneration of replacement heifers.

Known Benefits of Cross Breeding

Practitioners of animal husbandry are well aware that many advantagesare commonly observed with crossbreeding, are well documented, and canhave a big impact on net return. Heterosis (hybrid vigor) and breedcomplementarity are the primary benefits realized from a properlyplanned crossbreeding program. Specifically, heterosis refers to theincrease in performance or function observed in crossbreed hybridprogeny that is above what is expected based on the parents of theoffspring. Breed complementarity allows a breeder to capitalize on thestrengths of different breeds because no single breed excels at all ofthe traits that affect profitability.

Heterosis can be observed with respect to any health or productiontraits if there is sufficient genetic difference between the two breedsbeing crossed. If Breed 1 is crossed with Breed 2 the added benefitbeyond the average of both breeds' traits witnessed in the offspring isthe heterosis effect. The heterosis effect can be variable betweenbreeds and also across traits that are affected.

Maternal heterosis is the advantage realized by using a crossbred cowversus a straight-bred cow. Research has shown that crossbred hybridcows can have many advantages, which can include, for example, highercalving rate (increases of about 6% are commonly reported), higher calfsurvival rate (increases of about 4% are commonly reported), higher calfweaning weight (increases of about 5% are commonly reported), higherpost-weaning calf gain (increases of about 6% are commonly reported),higher milk production (increases of about 6% are commonly reported),higher efficiency (increases of about 8% are commonly reported),increased longevity (increases of about 38% are commonly reported), andincreased lifetime productivity (increases of about 23% are commonlyreported). Those skilled in the art are aware that presence or level ofany particular benefit can vary greatly in different crosses, and aregenerally believed to be significantly influenced by breed.

The goal of most commercial cow-calf producers is to increaseprofitability. While it is appreciated that using crossbreeding can havea significant positive impact on various aspects of production in adairy farm, including healthier and more fertile offspring as well asthe ability to introduce genetic diversity into the herd, it is alsounderstood that conventional crossbreeding strategies can produce cattleof inferior, instead of superior, quality or with unexpected negativetraits. One of the challenges encountered with traditional crossbreedfarming is that crossbreeds include the potential for unpredictablebehavioral traits, such as temperamental dispositions; such undesirabletraits can be amplified if crossbred hybrid animals (i.e., “F1” progeny)are subsequently in-bred or back-crossed in an effort to continue aline.

The present disclosure appreciates that crossbred herds are difficult tomaintain. That is, there is a risk of losing high performing traits ofF1 cattle. For example, if farmers try to maintain an F1 herd usingconventional approaches (e.g., as depicted in FIG. 1), they willtypically mate females of the herd to the F0 sire (see FIG. 1, leftpanel). However, loss of hybrid vigor with respect to particulardesirable typically results from such backcrossing. Such backcrossingcan also result in increased heterogeneity in the next generation ofanimals due to independent assortment of alleles in the parents. Analternative strategy to cross breeding has been to introduce a thirdbreed as the mating sire for the F1 hybrid animals (see FIG. 1, rightpanel). While this strategy has higher relative heterosis compared tothe backcrossing strategy it still suffers from the problem ofindependent assortment that results in unpredictable and highly variedphenotypes. The present disclosure, among other things, encompasses theinsight that there is currently a problem with crossbreeding systems inthat the initial, high performing and uniform F1 hybrid animals cannotbe used to make similarly high performing and uniform replacements withnatural service or artificial insemination due to segregation of allelesin the production of gametes from the F1 parent.

Inventive Strategies for Generating and/or Maintaining F1 CrossbreedDairy Cattle

The present disclosure encompasses the insight that, while dairyproducers can already benefit from F1 genetics, dairy herds wouldbenefit from an ability to support the production of their replacementfrom pregnancies occurring in the normal course of their dairyoperation. Pregnancies produced on hybrid cattle with current methods(artificial insemination or natural service) result in unpredictableoffspring due to recombination during the natural processes that createhaploids (eggs) in the hybrid animals. The present disclosure providesnew approaches that permit production and/or maintenance of valuable F1crossbreed dairy cattle herds.

Among other things, the present invention encompasses the recognitionthat an improved system for providing and/or maintaining cross-breddairy cattle herds involves identifying, characterizing, and/orselecting one or both F0 individuals or lines based on performance oftheir hybrid progeny.

As discussed herein, the superiority of hybrid animals (aka “F1cross-breeds”) has been documented in the academic literature fordecades. Despite evidence of superiority, cross breeding systems and useof F1s specifically constitute a small percentage of total dairyoperations. The present disclosure encompasses the insight that, wherecross breeding systems fail is in the production of the next generationof replacements. The F1 animal itself is a composite that has 50% of itsgenome from Breed A and 50% from Breed B. Its phenotype and performanceis therefore consistent. With natural or AI delivery the next animalproduced (the replacement) from this female presents challenges. Due tosimple Mendelian principles of independent assortment the Breed A andBreed B contributions will recombine in the production of haploids forthe next generation, and while on aggregate these haploids will be 50% Aand 50% B the actual contribution will be a normal distribution centeredat 50% A:B and approach 100% at the tails. Since every replacementpregnancy will be a single draw from this distribution you will notlikely get the middle AND every draw will get different genesrepresented from A or from B. Thus the haploid oocytes produced even ifmated to a SINGLE SIRE will differ markedly from each other inphenotype, performance, and even simple cow design. For this reason theusual end of a crossbreeding operation is a return to one of theoriginal F0 parent breeds through a system of recurrent backcrossing, orthrough simple acquisition of purebred animals from outside theoperation (see FIG. 1 and FIGS. 2A-2D). A business system that couldsustainably deliver hybrid F1 genetics, while generating the pregnanciesrequired for sustained milk production has potential for wide adoptionand positive impact throughout the dairy industry.

In accordance with the present disclosure, a system to deliver thisvalue can be assembled from the following components. (1) A two linebreeding system incorporating one or more: (a) male line(s)—which aredeveloped from current elite dairy breeds to provide semen and aredeveloped in a manner similar to the development of current AI sireswith the exception that genetic selection is driven by performance(predicted and measured) of the hybrid offspring; (b) femaleline(s)—which are also developed from current elite dairy breeds toprovide oocytes to be used in WF. These lines are developed with indexedbased genetic selection based on sustained female performance (in termsof oocytes yield and diary performance) and performance of the F1offspring; (c) a bull stud to maintain the male lines and produce semen;(d) an oocyte farm and female nucleus that provides both oocyteproduction for creation of F1 embryos and allows continuous geneticimprovement of the female line(s). (2) A testing system that allows highquality performance information to be obtained from F1 animals in highquality commercial dairy operations to provide: (a) testing of F1combinations of existing genetics to allow launch products to beperformance tested; (b) selection data to be obtained so the male andfemale line selections can be supported with F1 information and candiverge from breed dependent selection. (3) An IVF and embryo transfer(ET) service business that can support IVF in proximity to the oocytesfarm and ET services to dispersed commercial dairy operations. (4) Acustomer facing sales force that can sell new genetic products based onperformance differentiation. See FIG. 3.

In some embodiments, generating and/or maintaining high performinghybrid F1 dairy cattle involves use of modern reproductive technologiessuch as IVF, ET, artificial insemination, cryopreservation, and nucleartransfer among others. Such modern techniques have been used inindustries such as race horse breeding, where pure-bred animals arequite valuable, but not in the dairy industry [for the production ofcrossbreeds], where individual commercial production animals (i.e.,females) are typically considered to be of insufficient individual valueto warrant use of such technologies.

In some embodiments, the present invention encompasses the use of moderntechnologies such as IVF, ET, artificial insemination, cryopreservation,and nuclear transfer in the screening, generation, and maintenance ofhybrid dairy cattle.

Furthermore, in some embodiments, the present invention providestechnologies through which gamete samples from an F0 sire are mated to afirst plurality of female gametes from a first F0 dam, and also to asecond plurality of female gametes from a second F0 dam, different fromthe first F0 dam, but optionally of the same line as the first F0 dam).In many embodiments, each of the first and second F0 dams is of adifferent breed from the F0 sire. Traits of hybrid progeny from each ofthese first and second (and subsequent) crossbreed crosses are assessed,and the F0 sire (and optionally one or more of the F0 dams or dam lines)is selected for use in subsequent cross-breed matings based on desirableperformance of F1 progeny from the first and/or second crossbreedcrosses.

In some embodiments, the quality of hybrid progeny traits is assessedrelative to that of a reference animal. In some embodiments, the qualityof hybrid progeny traits is assessed relative to that of hybrid progenyof comparable cross-breed cross (optionally historical or simultaneous).In some embodiments, the quality of hybrid progeny traits is assessedwith reference to a crossbreed index or other industry standard.

In some embodiments, the present invention provides for continual[generation on generation] improvement of hybrid herds due to ongoingmonitoring of performance of hybrid crossbreed progeny traits, andcomparison with those of hybrid crossbreed progeny of other dairy cattlecrosses, so that periodic or continual improvements can be made inselection of F0 breeding stock.

In some embodiments, F1 embryos may be shipped from a first farm that isa stud farm to a second farm that is a gestational dam farm having aherd of gestational cows into which semen or embryos can be delivered.In some embodiments, F1 embryos can be shipped to a plurality ofdifferent gestational farms, so that a plurality of F1 progeny herds isproduced at the plurality of farms. In some embodiments, informationregarding F1 traits is provided by the gestational farm(s) (or bypurchasers of one or more of the F1 progeny cattle) to the stud farm sothat quality of the sire as breeding stock for hybrid dairy cattle canbe assessed. Analogously, in some embodiments, information regardinghybrid traits is provided to one or more of the gestational farm(s)and/or to the source farm of the F0 gamete used to generate the F1embryos so that quality of the dam or dam line as dairy cattle breedingstock can be assessed.

In some embodiments, assessment of F1 traits in progeny of one or morematings of gametes from particular F0 individuals or lines may beassessed at multiple points in time so that quality of F0 individuals asdairy cattle breeding stock can be periodically or continuallyreassessed. In some embodiments, one or more particular F0 individualsmay be de-selected over time as dairy cattle breeding stock.

Inventive Selection of Breeding Stock/Hybrid Breeding Indeces

In some embodiments, in accordance with the present invention, F0 cattlemay be selected based upon the performance of hybrid cattle that resultfrom cross-breed matings of their gametes. For example, the milkproduction performance of hybrid cattle can be evaluated, and F0 cattlemay be selected for use as breeding stock for a particular cross (e.g.,for a cross-breed cross) based on the performance of their F1 progenyfrom prior cross-breed crosses.

Alternatively or additionally, hybrid traits that may be evaluated mayinclude, for example, age at first calving, body depth, cell counts, cowconception rate, dairy form, daughter calving ease, daughter pregnancyrate (the rate at which F1 animals become pregnant with daughters),daughter still birth, fat pounds, milk fat percent feet and legs score,fertility, final score, foot angle, fore udder attachment, front teatplacement, heifer conception rate, ketosis, lameness rate and/or degree,locomotion, milk productive life, milking speed, milk protein percent,protein pounds, rear legs rear view, rear legs side view, rear teatplacement, rear udder height, reproductive life, resistance to cold,resistance to disease (e.g., mastitis, metritis, etc.), rump angle, rumpwidth, somatic cell score, sire calving ease, sire still birth, size,stature, strength, teat length, udder cleft, udder conformation, udderdepth, and combinations thereof.

Hybrid traits may be evaluated on the basis of phenotype, genotype, orboth, using any available techniques.

One or more hybrid traits may be utilized in one or more hybrid indexes;such hybrid indexes may facilitate organization, evaluation, and/orranking of hybrid cattle, which in turn may facilitate selection of F0individuals or lines based on performance of their hybrid progeny. Insome embodiments, relative weights are assigned to individual traits(e.g., within an index).

In some embodiments, milk production is deemed the most influential(e.g., heavily weighted) hybrid trait considered for selection of F0individuals or lines. In some embodiments, one or more of infertility,high somatic cell counts, and lameness is assigned a negative weight(e.g., is selected against) in an inventive method or index.

Among other things, technologies provided by inventive strategiesdescribed herein are expected to maximize milk production and/or tominimize turn-over or replacement rate in hybrid herds.

In some embodiments, reduced susceptibility to disease may be a keyperformance trait for evaluating hybrid cattle. Mastitis is an infectionof the udders that negatively impacts milk production. Selecting againstmastitis can improve the milk producing capabilities of the hybridcattle. High somatic cell counts are also an indicator of an infectionlike mastitis. Selecting against high somatic cell counts (SCC) canlower the incidences of disease.

Fertility is one of the major factors affecting the efficiency of anydairy herd. It can account for one of the major costs of production andalso represents an area where significant improvements can be made. Poordairy herd fertility is recognized as having many consequences, bothdirect and indirect. Most importantly: loss of milk production throughtoo many dry days or peak yield traded for later lactation yield;disruption to the calving season and milk production pattern; loss ofmature animal milk yields through early culling; extra veterinary costs;reduced calf sales; additional AI costs; enforced culling resulting inmore replacements being reared or bought; loss of valuable genetics; andlinkage with other problems such as nutritional imbalances andproduction shortfalls. The first step in reducing unnecessary fertilitylosses is detailed assessment of individual herd fertility performanceas well as selecting for F0 that will improve or maintain desiredfertility.

In some embodiments, desirable hybrid traits may include potentialsuitability as a beef cow. Dairy females that are no longer able toproduce milk are traditionally culled in favor of replacement females.Traditionally, dairy cows do not often provide meat of a quality in linewith beef cattle, sometimes leading to reduced value for the cattle.Having traits desirable to the beef markets would provide another sourceof profitability to dairy farmers. In some embodiments, dairy cattlecross-bred with beef cattle have traits desirable to the beef industrywithout sacrificing traits valuable to milk production.

In some embodiments, gametes from breeding stock males and/or femalesselected based on performance of hybrid progeny resulting from priorcrossbreed crosses are utilized to produce new hybrid embryos and/orhybrid progeny. In some embodiments, such gametes are collected forstorage (e.g., freezing/cryopreservation). In some embodiments, suchgametes are sold, for example to one or more farms or farmingfacilities.

In some embodiments, further breeding progress is obtained where bothsire and dam lines contribute to the F1 performance by selecting traitsseparated between the two lines. For example, in a hybrid animalresulting from a combination of Holstein and Jersey breeds, where theHolstein is the sire line and the Jersey is the dam line, the Holsteinparent can be selected with a focus on fluid milk production, whileignoring milk components and the Jersey parent can be selected with afocus on milk solids (e.g. fat and protein), for example optionallyignoring fluid milk production. In this way, further genetic progresscan be made than by trying to improve both traits in each of the parentlines.

Thus, the present disclosure provides selection strategies for breedingcattle that differ from those used in conventional dairy cattlehusbandry. Those skilled in the art, reading the present disclosure,will appreciate that it therefore also provides novel breeding indicesfor cattle breeds that are specifically tailored for generating qualityhybrid progeny (such indices may be referred to herein as “hybridbreeding indices”). For example, with respect to the particular casedescribed in the prior paragraph, the present disclosure provides ahybrid breeding index for a Holstein parent to be used in aHolstein/Jersey cross that focuses on milk production and not milkcomponents; and also provides a hybrid breeding index for a Jerseyparent to be used in such a cross that focuses on milk solids and notmilk production. Those skilled in the art will further appreciate that,in accordance with the present disclosure, different hybrid breedingindices may be utilized for a given strain or line depending on thepartner strain or line to be utilized in the cross. The presentinvention therefore provides sets of hybrid breeding indices for dairycattle breeds or lines, that may differ depending on the partner breedor line to be utilized in the cross.

Those skilled in the art, reading the present disclosure, willappreciate that one feature and implication of inventive technologies isthat breeding stock sires (or sire lines) and dams (or dam lines) areselected notwithstanding their own particular traits. That is, inaccordance with the present invention, it is not necessary that a cow bea high milk producer herself to act as a dairy cow breed stock dam forone or more F1 cross-breed progeny as described herein. The presentinvention, therefore provides technologies that, exactly contrary toconventional wisdom, select and/or utilize as dairy cow individualswhose personal dairy production trait or traits may be profoundlysub-par (e.g., relative to breed norms, standards, indexes, etc.), andin particularly may be materially below that/those of its F1 cross-breedprogeny.

Provided Herds

In some embodiments, the present invention provides for the creationand/or maintenance of high performing herds of cattle based upon desiredtraits. In some embodiments, herds can be generated through selection ofF0 sires/sire lines and dams/dam lines as determined by the performanceof hybrid cattle resulting from theses crosses. F0 cattle can bere-paired to generate new hybrid cattle with the desired traits.Techniques such as estrous synchronization and/or artificialinsemination can aid the breeding of F0 cattle to more efficientlyproduce high performing hybrid cattle.

Alternatively or additionally, in some embodiments, herds of highperforming hybrid cattle can be generated through embryonicmanipulation. Gametes from F0 cattle can be collected and used togenerate new hybrid cattle through in vitro fertilization and embryonictransfer. Gametes as well as fertilized embryos can be frozen and storedfor later implantation to build up a herd of high performing hybridcattle. In addition, sexed sperm can be used to favor the generation offemales for replacement cattle or more milk producers within the herd.

Herds of cross-bred cattle can be produced from breeds of cattleincluding but not limited to: Holstein, Guernsey, Ayrshire, Red andWhite, Milking Shorthorn, Jersey, Brown Swiss, Norwegian Red, BelarusRed, Belted Galloway, Angus, Beefmaster, Black Hereford, Brahman,Brangus, Charolais, Florida Cracker, Hereford, Highland, Pineywoods, RedAngus, Santa Gertrudis, Simmental, Tajima, Texas Longhorn, Limosin,Wagyu, and any combination thereof.

Among other things, the present invention provides of hybrid cattle thatare progeny of matings between F0 individuals where at least one of theF0 individuals was selected based on one or more attributes of priorhybrid progeny from matings involving gametes of the same F0 individual(or of another F0 individual in the same line, particularly on the damside). The present invention also provides herds of such hybrid cattle,and also collections of F1 cross-breed embryos, which may becryopreserved or otherwise prepared for storage, including for long termstorage.

In some embodiments, provided hybrid herds are pregnant. In someembodiments, provided hybrid herds are pregnant with F1 hybrid embryos,e.g., that may have resulted from the same cross as that which generatedthe pregnant hybrid progeny, and/or from a repeat cross of the same F0individuals (or individuals from the same line). In some embodiments,provided hybrid herds are pregnant with hybrid embryos from a secondmating cross of the same breeds utilized in the first cross thatgenerated the pregnant hybrid, wherein the second mating cross isconsidered to be an improved cross relative to the first cross becauseone or both of the F0 individuals whose gametes were utilized in thesecond cross was selected via a process of continued hybrid performancemonitoring as described herein. In some embodiments, provided F1 herdshave excess gestational capacity as compared with that required tomaintain the herd; in some embodiments, some or all of the excesscapacity is utilized to gestate beef cattle.

Embryonic and Fertilization Technologies

Various techniques have been developed and refined to permit humans tocontrol and/or effect animal matings optionally without animalintercourse (e.g., natural service) or even animal contact.Representative such techniques include, for example, in vitrofertilization, artificial insemination, cryopreservation (freezing) ofgametes or embryos, induction of multiple ovulations, embryo transfer,sex determination of sperm or embryos, nuclear transfer, cloning, etc.

In vitro production of ruminant embryos is a three-step processinvolving oocyte maturation, oocyte fertilization, and in vitro culture.Only 30-40% of such oocytes reach the blastocyst stage, at which theycan be transferred to a recipient or frozen for future use. The qualityof the oocyte can dramatically impact the proportion of immature oocytesthat form blastocysts while the post-fertilization culture environmenthas a major influence on the quality of the blastocyst. In someembodiments, use of sperm of a specific gender in conjunction with invitro embryo production is a potentially efficient means of obtainingoffspring of the desired sex. Concerns regarding the use of sexed sementechnology include the apparent lower fertility of sorted sperm, thelower survival of sorted sperm after cryopreservation and the reducednumber of sperm that could be separated in a specified time period.Assessment of embryo quality is a challenge. Morphological assessment isat present the most popular method for embryo selection prior totransfer. Other non-invasive assessment methods include the timing ofthe first cleavage division which has been linked to developmentalability. Quantitative examination of gene expression is an additionalvaluable tool to assess the viability of cultured embryos. A substantialamount of evidence exists to demonstrate that the culture conditions towhich the embryo is exposed, particularly in the post-fertilizationperiod, can have perturbing effects on the pattern of gene expression inthe embryo with potentially important long-term consequences.

IVF is a technique in which the oocytes are extracted from a donor cowby a method of aspiration from the reproductive tract. Selected oocytesare then incubated for a period of 24 hours; this is call the maturationperiod. After maturation, the eggs are fertilized 18 to 22 hours afterthe co-culture has been made. The embryos stay in the medium until the7th day, when they are ready to be transferred. This technique has threemain advantages over conventional In Vivo embryo collection. With IVF,it is not necessary to superovulate the cows, nor is it necessary tosynchronize them. This is a major breakthrough since the donor cows arenot exposed to hormones that might compromise the reproductive soundnessof the animals, and they can be worked without prior preparation timefor the procedure. Embryo production averages about 30% of the oocytesharvested, although this quantity varies depending on the breed, thedonor cow, and also the mating. Another advantage with the IVF is thatthe animals can be aspirated every 20 days instead of every 60 as in InVivo embryo collection. The other advantage of IVF is that the animalscan be harvested at a very young age; this will create a major impact onbreeding selection since it reduces the generation interval for theanimals with a specific desirable trait.

Artificial insemination (AI) has been used to obtain offspring fromgenetically superior males for more than 200 years. Well known methodsto cryopreserve (freeze) and store semen have made AI accessible to morelivestock producers. In the same manner as cryopreservation of semen,embryo freezing allowed for the global commercialization of animals withhigh genetic qualities. Semen from bulls is especially amenable tofreezing and long-term storage. In the dairy industry, where largenumbers of dairy cows are managed intensely, AI is simple, economical,and successful. More than 60 percent of dairy cows in the United Statesare bred by AI. However, the situation is different for beef cattle,where breeding populations are usually maintained on range or pastureconditions. In the United States beef industry, AI accounts for lessthan 5 percent of inseminations.

Development of ET technology allows producers to obtain multiple progenyfrom genetically superior females. Fertilized embryos can be recoveredfrom females (also called embryo donors) of superior genetic merit bysurgical or nonsurgical techniques. The genetically superior embryos arethen transferred to females (also called embryo recipients) of lessergenetic merit. In cattle, efficient techniques can recover fertilizedembryos without surgery, but only one or sometimes two embryos areproduced during each normal reproductive cycle. To increase the numberof embryos that can be recovered from genetically superior females, theembryo donor is treated with a hormone regimen to induce multipleovulations, or superovulation.

The beef industry in the United States prefers male calves, which tendto have higher body weights and higher feed efficiency (compared tofemale or heifer calves) when placed in feedlots for the growing andfinishing stages of meat production. In contrast, the dairy industryprefers heifer calves, which will ultimately produce offspring and milkfor human consumption. Thus, methods are needed to determine the sex ofsperm or embryos so producers can control the sex of the offspring oftheir livestock.

Since the mid-1980s, technology has been developed to transfer thenucleus from either a blastomere (cells from early, and presumablyundifferentiated cleavage stage embryos) or a somatic cell (fibroblast,skin, heart, nerve, or other body cell) to an enucleated oocyte(unfertilized female egg cell with the nucleus removed). This “nucleartransfer” produces multiple copies of animals that are themselves nearlyidentical copies of other animals (transgenic animals, geneticallysuperior animals, or animals that produce high quantities of milk orhave some other desirable trait, etc.). This process is also referred toas cloning. To date, somatic cell nuclear transfer has been used toclone cattle, sheep, pigs, goats, horses, mules, cats, rabbits, rats,and mice.

The technique involves culturing somatic cells from an appropriatetissue (fibroblasts) from the animal to be cloned. Nuclei from thecultured somatic cells are then microinjected into an enucleated oocyteobtained from another individual of the same or a closely relatedspecies. Through a process that is not yet understood, the nucleus fromthe somatic cell is reprogrammed to a pattern of gene expressionsuitable for directing normal development of the embryo. After furtherculture and development in vitro, the embryos are transferred to arecipient female and ultimately result in the birth of live offspring.The success rate for propagating animals by nuclear transfer is oftenless than 10 percent and depends on many factors, including the species,source of the recipient ova, cell type of the donor nuclei, treatment ofdonor cells prior to nuclear transfer, the techniques used for nucleartransfer, etc.

The present disclosure demonstrates the effectiveness of combiningfertilization technologies with trait selection to improve performancein dairy cattle. The present disclosure appreciates that engineeredmating technologies such as in vitro fertilization, artificialinsemination, cryopreservation (freezing) of gametes or embryos,induction of multiple ovulations, embryo transfer, sex determination ofsperm or embryos, nuclear transfer, cloning, etc. have not heretoforebeen extensively utilized for the production of dairy cattle. Withoutwishing to be bound by any particular theory, the present inventorspropose that conventional wisdom has been that such techniques would notoffer significant benefit in the dairy cattle industry, at least not ofa nature or type that could justify their significant expense. Thepresent disclosure demonstrates, however, that such technologies canoffer significant, heretofore unappreciated benefits in the dairy cattleindustry. For example, the present disclosure establishes that suchtechnologies uniquely facilitate development, maintenance, surveillance,and/or improvement of F1 crossbreed dairy cattle herds.

In some embodiments, embryos generated as a result of F0 selection basedupon hybrid performance can be supplied to other farms and businesses,for example to permit them to generate hybrid progeny and/or herds. Insome embodiments, the present invention allows for a business method ofscreening hybrid cattle and recreating high-performing hybrid cattle byselective breeding using the F0 gametes of their parents.

EXEMPLIFICATION Example 1 Reproducing Additional Hybrid Cattle Based onan Individual High Performing F1 Bovine

The present Example demonstrates, among other things, the optimizationof performance traits of cattle by selecting F0 sires/sire lines anddam/dam lines based upon the performance of hybrids.

Hybrid dairy cattle resulting from breeding programs are screened forvarious performance traits. Screening involves advanced genetic-basedtesting as well as simple observation and analysis of each bovine's milkoutput and other performance traits. Those that have the best milkproduction, fertility, resistance to disease, and overall health arerecorded along with their ancestral lineage. Gametes from the F0 siresand dams that produced the high performing F1 bovine are collected andstored for generation of additional high performing F1 cattle. If theyare available, the F0 males and females themselves are used for furtherbreeding. Female calves that have reached sexual maturity are implantedwith a pre-fertilized embryo (generated from the gametes of the ideal F0sire and dam). To bias a pregnancy towards an either female or maleoffspring, spermatozoa are sexed. High-performing hybrid cattle areimplanted with embryos from their F0 parents to help produce additionalhigh performing sister and/or brother cattle as well as continue themilk production process.

Example 2 Creating a Herd of High-Performing Hybrid Cattle

Once high performing hybrid and their sires/sire lines and dams/damlines have been established, a herd of high performing cattle will becreated. The novel F1 cattle are screened and analyzed as in Example 1to determine which are high performers. The F0 gametes from the parentsof these new high performers are also used for subsequent F1 cattleproduction.

REFERENCES

-   1. Madalena et al. Evaluation of Strategies for crossbreeding of    dairy cattle in Brazil. J. Dairy Sci. 73 (7), 1887-1901 (1990).-   2. Schefers, J. M. and Weigel K. A. Genomic selection in dairy    cattle: Integration of DNA testing into breeding programs. Animal    Front. 2 (1), 4-9 (2012).-   3. Xue et al. Milk production and energy efficiency of Holstein and    Jersey-Holstein crossbred dairy cows offered diets containing grass    silage. J. Dairy Sci. 94 (3), 1455-1464 (2011).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

We claim:
 1. A method of maintaining an F1 hybrid crossbreed herd ofdairy cattle comprising steps of: implanting an F1 hybrid crossbreedembryo into each of a plurality of F1 female dairy cattle, wherein eachof the F1 female dairy cattle resulted from a first F1-selectedcrossbreed cross, wherein an F1-selected cross is one that involvesfertilization of an F0 female gamete with an F0 male gamete, wherein theF0 male and female gametes are from different breeds; and one or both ofthe F0 male and female gametes is from an F0 individual or line selectedbased on one or more improved performance characteristics of prior F1progeny generated in crosses of the same individual or line; whereineach implanted F1 hybrid crossbreed embryo results from a secondF1-selected crossbreed cross.
 2. The method of claim 1, wherein thesecond F1-selected crossbreed cross utilizes male F0 gametes from thesame individual utilized in the first F1-selected crossbreed cross. 3.The method of claim 1, wherein the second F1-selected crossbreed crossutilizes a male gamete from an individual or line different from thatutilized in the first F1-selected crossbreed cross.
 4. The method ofclaim 3, wherein the different individual is selected based on improvedperformance of its F1 progeny relative to those of the corresponding F0individual used in the first F1-progeny selected crossbreed cross. 5.The method of claim 4, further comprising steps of: assaying one or moreperformance characteristics of a plurality of F1 individuals resultingfrom a plurality of crosses that utilize F0 gametes from one or both ofthe F0 individuals or lines utilized in the first F1-selected crossbreedcross; comparing the assayed one or more performance characteristicswith those of F1 individuals resulting from a plurality of crosses thatutilize F0 gametes from the different F0 individual or line; andselecting a different individual or line for a breeding stock individualor line.
 6. The method of claim 1, wherein the second F1-selectedcrossbreed cross utilizes male F0 gametes and female F0 gametes from thesame individuals utilized in the first F1-selected crossbreed cross. 7.The method of claim 1, wherein the second F1-selected crossbreed crossutilizes male F0 gametes from the same line utilized in the firstF1-selected crossbreed cross.
 8. The method of claim 1, wherein thesecond F1-selected crossbreed cross utilizes female F0 gametes from thesame line utilized in the first F1-selected crossbreed cross.
 9. Themethod of claim 1, wherein the second F1-selected crossbreed crossutilizes female F0 gametes from the same individual utilized in thefirst FI-selected crossbreed cross.
 10. The method of claim 1, whereinthe second F1-selected crossbreed cross utilizes female gametes from anindividual or line different from that utilized in the first F1-selectedcrossbreed cross.
 11. The method of claim 3, wherein the different lineis selected based on improved performance of its F1 progeny relative tothose of the corresponding F0 line used in the first F1 progeny selectedcrossbreed cross.
 12. The method of claim 1, further comprisingpreparing said hybrid crossbreed embryo by in vitro fertilization andculture.
 13. The method of claim 1, further comprising preparing saidhybrid crossbreed embryo by in vivo fertilization and collection. 14.The method of claim 1, wherein said implanted hybrid crossbreed embryois a previously frozen embryo.
 15. The method of claim 1, wherein saidF1 hybrid cross-breed embryo is a cross-breed embryo selected from thegroup consisting of a Holstein/Norwegian Red hybrid, a Holstein/Jerseyhybrid, a Jersey/Norwegian Red hybrid, a Friesian/Danish Red hybrid, aBrown Swiss/Ayrshire hybrid, a Guernsey/Ayrshire hybrid, a MilkingShorthorn/Ayrshire hybrid, a Brown Swiss/Guernsey hybrid, aGuernsey/Milking Shorthorn hybrid, and a Milking Shorthorn/Brown Swisshybrid.
 16. The method of claim 1, wherein said F1 hybrid cross-breedembryo is an embryo of a Brahman F0 parent.
 17. The method of claim 1,wherein said performance characteristics comprise: milk production,longevity, semen production, age at first calving, body depth, cellcounts, cow conception rate, dairy form, daughter calving ease, daughterpregnancy rate, daughter still birth, fat pounds, fat percent, feet andlegs score, fertility, final score, foot angle, fore udder attachment,front teat placement, heifer conception rate, ketosis, lameness rateand/or degree, locomotion, milk productive life, milking speed, proteinpercent, protein pounds, rear legs rear view, rear legs side view, rearteat placement, rear udder height, reproductive life, resistance tocold, resistance to disease, rump angle, rump width, somatic cell score,sire calving ease, sire still bilih, size, stature, strength, teatlength, udder cleft, udder conformation, and udder depth.
 18. The methodof claim 1, wherein the F0 female gamete or the F0 male gamete is of adairy cattle breed selected from the group consisting of Holstein,Friesian, Norwegian Red, Danish Red, Brown Swiss, Guernsey, Ayrshire,Jersey, Red & White, Milking Shorthorn, Lineback, Dutch Belted, Burlina,Belarus Red, Canadienne, Carora, Danish Jersey, Frankeston Red, GermanBlack Pied, Brahman, Illawarra, and Meuse-Rhine-Issel.
 19. The method ofclaim 1, wherein the performance characteristic for selection is notshared by F0 individuals.
 20. The method of claim 1, wherein theperformance characteristic for selection is superior in F1 as comparedwith both F0 parents or F0 lines.